A remarkable property of cells is their ability to generate endogenous rhythms with extremely long periods in the 24 hr range. To express this property, cells contain a circadian oscillating mechanism coupled to an output system. Also, environmental information must be coupled to some element of the circadian oscillating mechanism to account for the normal regulation or entrainment of circadian rhythms. Our long-term goals are to understand the mechanisms of entrainment and generation of circadian oscillators. The objectives of the proposed research are to identify proteins that play a role in the circadian system and to discover the regulatory mechanisms that link the proteins together to form an oscillatory network. The isolated eye of Aplysia contains a circadian pacemaker that is regulated over two distinct entrainment pathways. Previously, we have used light and serotonin (5-HT) as probes to define elements of the entrainment pathways and to identify protein candidates for components of the circadian oscillator. The first aim of our research is to determine the function of proteins that are putative components of entrainment pathways or the oscillating mechanism. We will explore the function of important proteins by. obtaining amino acid sequences of peptides obtained from protein spots of preparative 2D-gels. Only after the functions of these proteins are known can precise models of oscillator components be proposed and tested. Recently, we identified one putative oscillator protein of Aplysia as a lipocortin-like protein. The second aim of our research is to investigate the role of this lipocortin-like protein in the circadian oscillator. First, we will test the hypothesis that the lipocortin-like protein regulates arachidonic acid metabolism and thus affects other oscillating elements through second messengers produced from arachidonic acid. Secondly, we will use antibodies of the lipocortin-like protein to localize the protein and study its variability. The third and fourth aims are to investigate the role of transcription and protein kinase activity in the regulation and generation of the circadian oscillator. The results of our experiments will identify important proteins in the circadian system and suggest relationships between the proteins. Also, the functions of several. putative protein components of the circadian system will be revealed. Finally, the importance of transcription, kinase activity, and arachidonic acid metabolism in circadian timing will be elucidated.